Isolation, purification, characterization, cloning and sequencing of N α-acetyltransferase

ABSTRACT

This invention is directed to N.sup.α -acetyltransferase with a molecular weight of about 180,000 daltons, said N.sup.α -acetyltransferase being composed of two subunit peptides having molecular weights of about 95,000 each, having enzyme activity greater than 100 wherein one unit of activity is defined as 1 pmol of acetyl residues incorporated into adrenocorticotropic hormone (ACTH) (amino acids 1-24) under standard assay conditions. This invention is further directed to a method for purifying the N.sup.α -acetyltransferase.

Cross-Reference to Related Applications:

This application is a continuation-in-part application of U.S. Pat. No.153,361, filed Feb. 8, 1988, now abandoned, the contents of which arefully incorporated herein by reference.

Field of the Invention

The invention is directed to the isolation, purification,characterization, cloning and sequencing of N.sup.α -acetyltransferaseand to the enzyme itself.

BACKGROUND OF THE INVENTION

An acetyl moiety was discovered as the amino-terminal blocking group ofviral coat protein in 1958 (Narita, K., Biochim Biophys. Acta 28:184-191(1958) and of hormonal peptide in 1959 (Harris J. I., Biochem. J.71:451-459 (1959). Since then, a large number of proteins in variousorganisms have been shown to possess acetylated amino-terminal residues.For example, mouse L-cells and Ehrlich ascites cells have about 80% oftheir intracellular soluble proteins N.sup.α -acetylated (Brown, J. I.and Roberts, W. K., J. Biol. Chem. 251:1009 (1976) and Brown, J. L. J.Biol. Chem. 254:1447 (1979)). In lower eukaryotic organisms, about 50%of the soluble proteins are acetylated (Brown, J. L., Int'l Conor.Biochem. Abstr. (Internation Union of Biochemistry, Canada) Vol. 11:90(1979)). These data demonstrate that N.sup.α -acetyl is a very importantblocking group. It has been suggested that the biological function ofthis blocking group may be to protect against premature proteincatabolism (Jornvall, H., J. Theor. Biol 55:1-12 (1975)) and proteinproteolytic degradation (Rubenstein, P. and Deuchler, J., J. Biol. Chem.254:11142 (1979)). However, in mouse L-cells such N.sup.α -acetylationdoes not apparently have this biological function (Brown, J. L., J.Biol. Chem. 254:1447 (1979)).

Although a clear general function for N-acetylation has not beenassessed with certainty, some specific effects for a small number ofproteins have been observed. Nonacetylated NADP-specific glutamatedehydrogenase in a mutant of Neurospora crassa is heat-unstable, incontrast to the acetylated form (Siddig et al., J. Mol. Biol. 137:125(1980)). A mutant of Escherichia coli, in which ribosomal protein S5 isnot acetylated, exhibits thermosensitivity (Cumberlidge, A. G. andIsono, K., J. Mol. Biol. 131:169 (1979)). N.sup.α -acetylation of two ofthe products from the precursor protein proopiomelanocortin has aprofound regulatory effect on the biological activity of thesepolypeptides; the opioid activity of β-endorphin is completelysuppressed, while the melanotropic effect of α-MSH is increased ifN.sup.α -acetylated (Smyth et al., Nature 279:252 (1970); Smyth, D. G.and Zakarian, S., Nature 288:613 (1980); and Ramachandran, J. and Li, C.H., Adv. Enzymol. 29:391 (1967)). Both acetylated and nonacetylatedcytoplasmic actin from cultured Drosophila cells participate in theassembly of microfilaments, the latter, however, with less efficiency(Berger et al., Biochem. Genet. 19:321 (1981)). More recently, the rateof protein turnover mediated by the ubiquitin-dependent degradationsystem was shown to depend on the presence of a free α-NH2 group at theN-terminus of a protein (Hershko et al., Proc. Nat'l Acad. Sci. U.S.A.81:9021-9025 (1984) and Bachmair et al., Science 234:179-186 (1986)),suggesting that N.sup.α -acetylation may have a role in impeding proteinturnover.

Given the importance of N-acetylation for the function and the abilityof these N-acetylated proteins to modulate cellular metabolism, it is ofinterest to examine the subcellular location, substrate specificity, andregulation of protein acetyltransferase. In order to cast light on thebiological implications of the acetylation and to elucidate theenzymatic mechanism of the reaction, as a first step, an enzyme must beisolated that is able to catalyze the aminoterminal acetylation in orderto investigate its substrate specificity. The existence of such anacetyltransferase has been demonstrated and studied in E. coli forribosomal protein L12 (Brot et al., Arch. Biochem. Biophys. 155:475(1973)), in rat liver (Pestana, A. and Pitot, H. C. Biochemistry 14:1404(1975); Green et al., Can. J. Biochem. 56:1075 (1978)); and Pestana, A.and Pitot, H. C. Biochemistry 14:1397 (1975)), calf lens (Granger etal., Proc. Nat'l Acad. Sci. U.S.A. 73:3031 (1976)), rat pituitary(Woodford, T. A. and Dixon J. E., J. Biol. Chem. 254:4993 (1979); Pease,K. A. and Dixon J. E., Arch. Biochem. Biophys. 212:177 (1981); andGlembotski, C. C., J. Biol. Chem. 257:10501 (1982)), ox pituitary(Massey D. E. and Smyth, D. G., Biochem. Soc'y Trans. 8:751-753 (1980),hen oviduct (Tsunasawa et al., J. Biochem. 87:645 (1980)), and in wheatgerm (Kido et al., Arch. Biochem. Biophys. 208:95 (1981)). However,isolation of this enzyme was not achieved, and only the enzyme from henoviduct has been partially purified about 40-fold (Tsunasawa et al., J.Biochem. 87:645 (1980). The inability to isolate and purify theseenzymes is due to their low concentration and extreme instability afterpurification.

SUMMARY OF THE INVENTION

According to this invention, an N.sup.α -acetyltransferase whichtransfers an acetyl group from acetyl coenzyme A to the N-terminal aminogroup of polypeptides was isolated and purified 4600-fold tohomogeneity. The invention therefore relates to the purification tohomogeneity of N.sup.α -acetyltransferase and to the purified N.sup.α-acetyltransferase.

The N.sup.α -acetyltransferase was purified by successive purificationsteps using ion exchange, hydroxylapatite, and affinity chromatography.The N.sup.α -acetyltransferase is composed of two identical subunits.The molecular weight (M_(r)) of the native enzyme was estimated to be180,000±10,000 daltons by gel filtration chromatography, and the M_(r)of each subunit was estimated to be 95,000±2,000 daltons by SDS-PAGE.The enzyme has a pH optimum near 9.0 and displays a maximum activity attemperatures from 30+ to 42° C. By chromatofocusing on Mono-P, theisoelectric point of this enzyme was determined to be 4.3. Among aseries of divalent cations, Cu²⁺ and Zn²⁺ were demonstrated to be themost potent inhibitors of the enzyme. By using several syntheticpeptides, it has been demonstrated that the enzyme has a definedsubstrate specificity dependent on the amino-terminal residue and thatthe enzyme may be involved in N-terminal processing of yeast proteins.

Further, the complete amino acid sequence for the yeast N.sup.α-acetyltransferase was deduced from cDNA. The N.sup.α -acetyltransferaseenzyme was cloned from a yeast λgt11 cDNA library and a full-length cDNAencoding yeast N.sup.α -acetyltransferase was sequenced. Southern blothybridizations of genomic and chromsomal DNA reveal that the enzyme isencoded by a single gene which is localized on chromosome IV. This geneis designated herein as "AAA1" (Amino-terminal Alpha-aminoAcetyltransferase 1). This yeast cDNA forms the basis for elucidatingthe biological function and regulation of N.sup.α -acetyltransferase ineukaryotic protein synthesis and degradation. Further, by usingsite-directed mutagenesis, the catalytic mechanism and regulation ofN.sup.α -acetyltransferase may be elucidated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chromatography of the yeast acetyltransferase onDEAE-Sepharose (0.2 M KCl). An aliquot of the supernatant from yeasthomogenates was chromatographed on DEAE-Sepharose (0.2 M KCl). Theeluant for A₂₈₀ was measured and acetyltransferase activity was assayedas described in the Examples. Fractions containing acetyltransferaseactivity were pooled as indicated by horizontal bar.

FIG. 2 shows the chromatography of the yeast acetyltransferase on DEAESepharose (0.05 to 0.5 M KCl). The acetyltransferase pool fromDEAE-Sepharose (0.2 M KCl) was concentrated, dialyzed, and an aliquotwas chromatographed on a DEAE-Sepharose (0.05 to 0.5 M KCl) and analysedfor A₂₈₀, conductivity, and acetyltransferase activity as described inthe Examples. Fractions containing acetyltransferase activity werepooled as indicated by horizontal bar.

FIG. 3 shows the chromatography of the yeast acetyltransferase onhydroxylapatite. The acetyltransferase pool from DEAE-Sepharose (0.05 to0.5 M KCl) was concentrated, dialyzed, and an aliquot waschromatographed on a hydroxylapatite column and analyzed for A₂₈₀,conductivity, and acetyltransferase activity as described in theExamples. Fractions containing acetyltransferase activity were pooled asindicated by horizontal bar.

FIG. 4 shows the chromatography of the yeast acetyltransferase on DE52cellulose. The acetyltransferase pool from hydroxylapatite wasconcentrated, dialyzed, and an aliquot was chromatographed on a DE52cellulose column and analyzed for A₂₈₀, conductivity, andacetyltransferase activity as described in the Examples. Fractionscontaining acetyltransferase activity were pooled as indicated byhorizontal bar.

FIG. 5 shows the chromatography of the yeast acetyltransferase onAffi-Blue gel.

FIG. 6 shows the estimation of the molecular weight of denatured yeastacetyltransferase by SDS-PAGE.

FIG. 7 shows the estimation of the molecular weight of native yeastacetyltransferase by gel filtration. The purified enzyme was applied toa Sepharose CL-4B column (2.5×96 cm). The apparent molecular weight ofnative yeast acetyltransferase was calculated from the relative elutionvolume of standard proteins to be 180,000±10,000 daltons. The elutionvolume was determined by absorbance at 280 nm and enzyme activity.

FIG. 8 shows chromatofocusing of yeast acetyltransferase on a Mono Pcolumn.

FIG. 9 shows the effect of temperature on yeast acetyltransferase.

FIG. 10 shows the specific activity of yeast acetyltransferasedetermined in 50 mM HEPES, potassium phosphate, CHES, and CAPS buffersof different pH.

FIG. 11 shows the HPLC separation of yeast N.sup.α -acetyltransferasetryptic peptides. Numbers refer to the tryptic peptides, the sequencesof which are shown in FIG. 12C.

FIG. 12 shows the cloning and sequencing of the cDNA encoding yeastN.sup.α -acetyltransferase. (A) shows oligonucleotide probes used forinitially screening the αgt11 library. The nucleotide positionsindicated by the asterisks differ from the actual DNA sequence shown in(C). The numbering of the tryptic peptides is as follows: the firstnumber refers to the corresponding peak in FIG. 11, the second numberrefers to the peak in the isocratic HPLC separation (data not shown),and the third number refers to the peak in the second isocratic HPLCseparation (data not shown). (B) shows the restriction map and DNAsequencing strategy for the cDNA clones. The arrows indicate thedirection and extent of sequence determination for each fragment afterexonuclease III deletion. (C) shows the nucleotide and deduced aminoacid sequence of N.sup.α -acetyltransferase cDNA clones.

FIG. 13 shows a hydrophobicity plot of yeast N.sup.α -acetyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

N.sup.α -acetyltransferase is an enzyme which transfers an acetyl groupfrom acetyl coenzyme A to the amino-terminal of a protein orpolypeptide. The structure of acetyl coenzyme A is given below: ##STR1##

I. Isolation, Purification, and Sequencing of N.sup.α-acetyltransferase.

In accordance with this invention, N.sup.α -acetyltransferase can beisolated from a sample containing the enzyme. Any sample that containsthe enzyme may be used as a starting material according to the methodsdescribed in this invention. N.sup.α -acetyltransferase appears to beubiquitous, being found in all eukaryotes, including plants, animals,and multicellular organisms. The enzyme is also believed to be presentin prokaryotes. Therefore, any source of N.sup.α -acetyltransferase iscontemplated in this invention. As used herein, the sample containingthe enzyme will be referred to simply as "sample," which is intended toinclude N.sup.α -acetyltransferase containing sample.

The isolation and purification of N.sup.α -acetyltransferase willhereinafter be described from a yeast sample, although it is to beunderstood that other samples could be used as the source material. Thepreferred method for purifying the N.sup.α -acetyltransferase of thepresent invention is that of Lee, F.-J. S., et al. (J. Biol. Chem.263:14948-14955 (1988), which reference is incorporated by referenceherein in its entirety).

Yeast cells were spheroplasted by lyticase and homogenized in hypotonicbuffer with a Dounce homogenizer. Yeast acetyltransferase was releasedfrom cells lysate into buffer B containing 0.2 M KCl by gently shaking.After centrifugation, the supernatant was concentrated byultrafiltration with PM-30 membrane and dialyzed overnight against HDGbuffer (20 mM HEPES-K⁺, pH 7.4, 0.5 mM DTT, 10% (v/v) glycerol and 0.02%NaN₃) containing 0.2 M KCl. The yeast acetyltransferase is not a stableenzyme and 10% of glycerol was used to extend the half-life of theenzyme.

The next step in the purification was removal of residual cellbiomaterials in the supernatant to avoid protein-biomaterialsaggregation in subsequent steps. Ion exchange can be used for thisprocedure. In this step, the ion exchange used was DEAE-Sepharosechromatography with constant salt (0.2 M KCl) elute which was found tobe most gentle procedure. About 80% of biomaterials or proteins wasremoved with a 107% recovery of yeast acetyltransferase activity. FIG. 1shows the chromatography of the yeast acetyltransferase onDEAE-Sepharose (0.2 M KCl).

Peak fractions from DEAE-Sepharose (0.2 M KCl) column were pooled,concentrated, dialyzed against HDG buffer containing 0.05 M KCl, andloaded onto a DEAE-Sepharose column with a continuous salt gradient(0.05 to 0.5 M KCl) elute. FIG. 2 shows the chromatography of the yeastacetyltransferase on DEAE-Sepharose (0.05 to 0.5 M KCl). A singlesymmetrical activity peak was generated which centered at 0.18 M KCl. Inthis step, a 204% recovery of the acetyltransferase activity wasachieved (Table 1), suggesting that an inhibitor was removed.

Peak fractions from DEAE-Sepharose (0.05 to 0.5 M KCl) were pooled,concentrated, dialyzed into 0.05 M potassium phosphate buffer, pH 7.4,p.5 mM DTT, 10% (v/v) glycerol, 0.02% NaN₃ and applied to an adsorptioncolumn using hydroxylapatite. As is known in the art, a hydroxylapatitecolumn will selectively adsorb proteins onto calcium ions in the calciumhydroxyphosphate packing. The hydroxylapatite column was eluted with alinear salt gradient and a single peak of activity at 0.36 M potassiumphosphate was generated. In this step the major peak of total proteinelution occurred at 0.5 to 2.0 M potassium phosphate. FIG. 3 shows thechromatography of the yeast acetyltransferase on hydroxylapatite.

Peak fractions from hydroxylapatite column were pooled, concentrated,dialyzed against HDG buffer containing 0.05 M KCl, and loaded onto anion exhange column, DE52-cellulose, with a continuous salt gradient. Asingle activity peak was generated which centered at 0.14 M KCl. FIG. 4shows the chromatography of the yeast acetyltransferase on DE52cellulose.

Peak fractions from DE52-cellulose column were pooled, concentrated,dialyzed against HDG buffer containing 0.05 M KCl, and loaded onto anaffinity column, Affi-Blue gel, with a continuous salt gradient (0.05 to1.0 M KCl) elute. A single activity peak was generated which centered at0.6 M KCl. FIG. 5 shows the chromatography of the yeastacetyltransferase on Affi-Blue gel column and analyzed for A₂₈₀,conductivity, and acetyltransferase activity as described above in"Materials and Methods." Fractions containing acetyltransferase activitywere pooled as indicated by horizontal bar.

Using this series of chromatography steps, yeast acetyltransferase waspurified approximately 4600-fold over the cell extract with a 27% yieldas seen in Table 1.

                  TABLE 1                                                         ______________________________________                                        PURIFICATION OF N-ACETYLTRANSFERASE                                           FROM YEAST                                                                                               Specific                                                                              Purifi-                                              Activity Protein Activity                                                                              cation                                                                              Yield                                Step      (Units)  (mg)    (Unit/mg)                                                                             (fold)                                                                              (%)                                  ______________________________________                                        1.  Crude     30200    17700 1.7     1.0   100                                    Extract                                                                   2.  DEAE-     32200    3710  8.7     5.1   107.sup.b                              Sepharose.sup.a                                                           3.  DEAE-     61500    1470  41.8    24.5  204.sup.b                              Sepharose.sup.c                                                           4.  Hydroxyl- 19300    53.6   360    210    64                                    apatite                                                                   5.  DEAE-     12700    8.58  1500    870    42                                    Cellulose                                                                 6.  Affi-Blue  8160    1.05  7800    4600   27                                    Gel                                                                       ______________________________________                                         .sup.a The results for step 2 are the combined yields of two                  chromatographies. Elution was 0.2 M KCl.                                      .sup.b An inhibitor was removed by this step                                  .sup.c Elution was 0.05 to 0.2 M KCl                                     

As used herein, the term "substantially pure" or "substantiallypurified" is meant to describe N.sup.α -acetyltransferase which issubstantially free of any compound normally associated with the enzymein its natural state, i.e., free of protein and carbohydrate components.The term is further meant to describe N.sup.α -acetyltransferase whichis homogeneous by one or more purity or homogeneity characteristics usedby those of skill in the art. For example, a substantially pure N.sup.α-acetyltransferase will show constant and reproducible characteristicswithin standard experimental deviations for parameters such as thefollowing: molecular weight, chromatographic techniques, and such otherparameters. The term, however, is not meant to exclude artificial orsynthetic mixtures of the enzymes with other compounds. The term is alsonot meant to exclude the presence of minor impurities which do notinterfere with the biological activity of the enzyme, and which may bepresent, for example, due to incomplete purification.

II. Identification, Purification, and Characterization of N.sup.α-acetyltransferase. Molecular Weight.

SDS-polyacrylamide gels of the purified sample reveal a single band whenstained with Coomassie blue. The electrophoresis was performed accordingto the method of Laemmli, U. K., Nature 227:680-685 (1970) using a 8%gel. The gel was stained with Coomassie blue. Standard proteins of 45,66, 97, 116, and 205 molecular weight were run with the crude extract,DEAE-Sepharose pool, DEAE-Sepharose pool, hydroxylapatite pool,DEAE-cellulose pool and Affi-Blue-gel pool. SDS-polyacrylamide gelelectrophoresis showed that the purified enzyme has a molecular weightof 95,000±2,000 daltons. FIG. 6 shows the estimation of the molecularweight of denatured yeast acetyltransferase by SDS-PAGE. The molecularweight of denatured yeast acetyltransferase was calculated by usingstandard proteins. Gel filtration chromatography on Sepharose CL-4Bshowed that the native molecular weight of the acetyltransferase isapproximately 180,000 daltons. FIG. 7 shows the estimation of themolecular weight of native yeast acetyltransferase by gel filtration.These data suggest that the yeast acetyltransferase is composed of twoidentical subunits. Purified yeast acetyltransferase was subjected toamino acid analysis. The results of this analysis are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        AMINO ACID COMPOSITION OF                                                     N-ACETYLTRANSFERASE FROM YEAST.sup.a                                          Amino Acid   Calculated Residues.sup.b                                        ______________________________________                                        Asx          103                                                              Thr          24                                                               Ser          43                                                               Glx          96                                                               Pro          32                                                               Gly          39                                                               Ala          61                                                               Val          41                                                               Met           3                                                               Ile          41                                                               Leu          106                                                              Tyr          32                                                               Phe          45                                                               Lys          73                                                               His          12                                                               Arg          37                                                               ______________________________________                                         .sup.a Purified acetyltransferase was obtained from gel elution after         SDSPAGE.                                                                      .sup.b Residues per subunit of enzyme were calculated on the basis of a       molecular weight of 95,000. No correction was made for the amounts of the     amino acids ser and thr that are destroyed during the hydrolysis. Cys and     trp were not determined by this method.                                  

Biochemical Properties of N.sup.α -acetyltransferase.

Chromatofocusing Mono P was used to determine the pI ofacetyltransferase. One activity peak was observed at pH 4.3. FIG. 8shows the chromatofocusing of yeast acetyltransferase on a Mono Pcolumn. The partial purified enzyme from DE52 column was applied to MonoP column equilibrated with 25 mM Tris-Bis buffer (pH 6) and eluted byPolybuffer 74 (pH 3.6) at the flow rate of 1 ml/min at 4° C. Elution wasmonitored by the absorbance at 280 nm, and 0.5 ml fraction werecollected for measurement of pH and enzyme activity.

FIG. 9 shows the effect of temperature on yeast acetyltransferase. FIG.10 shows the specific activity of yeast acetyltransferase was determinedin 50 mM HEPES, potassium phosphate, CHES, and CAPS buffers of differentpH's. The temperature optimum for yeast acetyltransferase was determinedby assaying under standard conditions. Assays were performed from 5° to55° C. as indicated in FIG. 9. The yeast acetyltransferase displays amaximum activity at temperatures from 30° to 42° C. Irreversibledenaturation occurred after 1 minute at 65° C. The pH dependence ofyeast acetyltransferase was measured by assaying at pH values from 5 to11 in the presence of 50 mM HEPES, potassium phosphate, CHES, and CAPSbuffers as indicated in FIG. 10. Maximum enzyme activity occurred at pH9.0. Enzyme activity was less than 25% below pH 6.5 and above pH 11.Addition of KCl or NaCl up to 0.45 M concentration did not affect theenzyme reaction. A 50% reduction in enzyme activity was observed whenthe assay was performed at 0.6 M KCl or 0.6 M NaCl.

Effect of Divalent Cations on Enzyme Reaction.

The effect of various divalent cations on the enzyme activity wasdetermined as shown in Table 3. At 1 mM concentration, Ca²⁺, Mg²⁺ had noeffect, whereas nearly complete inhibition occurred in the presence atMn²⁺, Fe²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺. Cu²⁺ and Zn²⁺ were the most potentinhibitors. It was verified that the observed effects of metal ions werenot due to the SO₄ ⁻² anion.

                  TABLE 3                                                         ______________________________________                                        EFFECT OF DIVALENT CATIONS ON                                                 ENZYME ACTIVITY.sup.a                                                                 Activity (%)                                                                  Concentration (mM)                                                    Addition  1             0.1    0.01                                           ______________________________________                                        None      100           --     --                                             CaCl.sub.2                                                                              100           --     --                                             MgCl.sub.2                                                                              99            120    --                                             MgSO.sub.4                                                                              120           120    --                                             MnCl.sub.2                                                                              28            43     84                                             CoCl.sub.2                                                                              20            60     86                                             CdCl.sub.2                                                                              12            42     90                                             FeSO.sub.4                                                                              12            53     78                                             CuSO.sub.4                                                                               0             0     58                                             ZnSO.sub.4                                                                               0             0     40                                             ______________________________________                                         .sup.a Yeast acetyltransferase was incubated in the presence of various       divalent cations at a room temperature for 5 min. The enzyme activity was     determined by the addition of substrate directly to the incubation mixtur     followed by the standard assay procedure.                                

Effect of Chemical Modifications on the Enzyme Reaction

To evaluate the possible catalytic role of amino acid residues in theacetylation reaction of this enzyme, several chemical modifications werecarried out (Table 4). Yeast acetyltransferase was incubated with eachreagent at 30° C. for 15 minutes, dialyzed against 50 mM HEPES buffer,pH 7.4, 150 mM DTT at 4° C. for 3-4 hours. The enzyme activity wasdetermined as described above. The reaction of acetyltransferase with 1or 5 mM diethyl pyrocarbonate, a histidine-modifying reagent, caused acomplete inactivation of the enzyme. The reagents used in Table 4 are:reagents which modify cysteine and cystine residues (NEM,N-ethylmaleimide; IAA, iodoacetic acid; IAM iodoacetamide; pCMB,p-chloromercuribenzoate; DTT, dithiothreitol); reagents which modifyHIS, TYR or LYS residues (DEPC, diethyl pyrocarbonate); reagents whichmodify HIS, TYR or TRP residues (NBS, N-Bromosuccinimide); reagentswhich modify HIS or carboxyl (Woodward's K; N-ethyl-5-phenylisoxazolium3'sulfonate); reagents which modify lysine or primary amino acidresidues (Succinic anhydride; TNBS, 2,4,6-trinitrobenzenesulfonic acid);reagents which modify TYR residues (N-acetylimidazole); reagents whichmodify TRP residues (HNBS(CH₃).sub. 2 -Br;dimethyl-(2-hydroxy-5-nitrobenzyl) sulfonium bromide); reagents whichmodify SER residues (PMSF); and reagents which chelate metal ions(DEAE).

                  TABLE 4                                                         ______________________________________                                        Effect of Protein Modification Reagants on Enzyme Activity                    of N-alpha-Acetyltransferase from S. cerevisiae                                                           Enzyme Activity                                   Reagant Added Concentration (mM)                                                                          (%)                                               ______________________________________                                        None                        100                                               NEM           1.0           92                                                              10.0          13                                                IAA           1.0           100                                                             5.0           73                                                IAM           1.0           98                                                              10.0          63                                                pCMB          1.0           100                                                             10.0          55                                                pCMB + DTT    1.0 + 10.0    100                                                             10.0 + 60.0   160                                               DTT           1.0           100                                                             10.0          110                                                             50.0          120                                               DEPC          0.5           52                                                              1.0           1.6                                                             5.0           0                                                 DEPC + Hydroxlamine                                                                         1.0 + 250.0   0                                                               1.0 + 500.0   0                                                 NBS           0.5           30                                                              5.0           0.5                                               Woodward's K  1.0           79                                                              10.0          0                                                 Succinic Anhydride                                                                          1.0           94                                                              10.0          71                                                TNBS          1.0           94                                                              10.0          76                                                N-acetylimidazole                                                                           1.0           100                                                             10.0          63                                                HNBS(CH.sub.3).sub.2 -Br                                                                    1.0           82                                                              10.0          70                                                PMSF          1.0           140                                                             10.0          134                                               DEAE          2.5           120                                                             25.0          135                                               2-Mercaptoethanol                                                                           10.0          110                                               ______________________________________                                    

Substrate Specificity.

A partial determination of substrate specificity of yeastacetyltransferase was carried out using a substrate containing the first24 amino acid residues of adrenocorticotropic hormone (ACTH). The aminoacid sequences of all ACTH substrates is as described by Lee, F.-J.S.,et al. (J. Biol. Chem. 263:14948-14955 (1988), which reference has beenincorporated by reference herein in its entirety).

Table 5 shows that the acetylation efficiency of the [Phe² ] analoguewas not significantly different from ACTH (amino acids 1-24). Fourtruncated forms of ACTH (amino acids 4-10), (amino acids 11-24), (aminoacids 7-38) and (amino acids 18-39), lacking different N-terminalresidues of ACTH, were not acetylated. β-Endorphin can be acetylated atN-terminal Tyr residue by rat pituitary acetyltransferase but not byyeast acetyltransferase (Table 6). The substrate specificity usingnatural substrates in the yeast was further investigated. Yeast alcoholdehydrogenase (ADH) is naturally acetylated at its N-terminal Serresidue. Human superoxide dismutase (SOD) is naturally acetylated at itsN-terminal Ala residue and also as expressed as a recombinant protein inyeast. However, endogenous yeast SOD, which is 48% identical to humanSOD, is not N-acetylated. Whether or not N-terminal sequence differencesbetween these proteins account for the differences in acetylationremains unclear. As shown in Table 6, synthetic yeast ADH (amino acids1-24) and synthetic human SOD (amino acids 1-24) can be acetylated bythis enzyme. However, yeast ADH already naturally acetylated at itsN-terminal Ser residue or synthetic yeast SOD (amino acids 1-24) cannotbe acetylated. In addition, yeast enolase containing an Ala residue witha free α-NH₂ group is known not to be N-acetylated in vivo and indeedcannot be acetylated by this enzyme. Furthermore, neither calf thymuslysine- or arginine-rich histones could be acetylated by the yeastacetyltransferase.

                  TABLE 5                                                         ______________________________________                                        RELATIVE ACTIVITY OF YEAST                                                    ACETYLTRANSFERASE FOR THE N-ACETYLATION OF                                    ACTH FRAGMENTS                                                                Substrate        Activity (%)                                                 ______________________________________                                        ACTH (1-24)      100 ± 5                                                   [Phe.sup.2 ]ACTH (1-24)                                                                         90 ± 9                                                   ACTH (4-10)      0                                                            ACTH (11-24)     0                                                            ACTH (7-38)      0                                                            ACTH (18-39)     0                                                            ______________________________________                                         Data reported as mean activity ± SD (N = 3-5).                        

                  TABLE 6                                                         ______________________________________                                        RELATIVE ACTIVITY OF YEAST                                                    ACETYLTRANSFERASE FOR THE                                                     N-ACETYLATION OF VARIOUS                                                      SYNTHETIC PEPTIDES AND NATIVE PROTEINS                                        Substrate                 Activity (%)                                        ______________________________________                                        ACTH (1-24) (Human)       100 ± 5                                          β-ENDORPHIN (Human)  2 ± 2                                            ALCOHOL DEHYDROGENASE (1-24) (Yeast)                                                                    92 ± 8                                           ALCOHOL DEHYDROGENASE (Yeast)                                                                           4 ± 2                                            SUPEROXIDE DISMUTASE (1-24) (Yeast)                                                                     0                                                   SUPEROXIDE DISMUTASE (1-24) (Human)                                                                     86 ± 6                                           ENOLASE (1-24) (Yeast)    4 ± 2                                            HISTONE (lysine-rich) (calf thymus)                                                                     0                                                   HISTONE (arginine-rich) (calf thymus)                                                                   0                                                   ______________________________________                                         Data reported as mean activity ± SD (N = 3-5).                        

Amino Acid Sequences of N.sup.α -acetyltransferase.

The N.sup.α -acetyltransferase was cleaved with trypsin and thefragments were separated on an HPLC phenyl reverse phase column. Peptidefagments (indicated with dashed lines in FIG. 12) were sequenced. Thesequence information from two of the sequenced peptide fragments,Fragment 15-3-1 and Fragment 29-1 are as follows:

Fragment 15-3-1:gly-val-pro-ala-thr-phe-ser-asn-val-lys-pro-leu-tyr-gln-arg

Fragment 29-1:phe-ile-pro-leu-thr-phe-leu-gln-asp-lys-glu-glu-leu-ser-lys

As detailed below, the sequence information from these two peptidefragments may be used to synthesize oligonucleotide probes as describedin Example 2.

III. Genetic Engineering of N.sup.α -Acetyltransferase.

Sequencing of N.sup.α -acetyltransferase.

The inventors have Completed the molecular cloning and determined thecomplete cDNA sequence analysis of a eukaryotic N.sup.α-acetyltransferase gene. The yeast N.sup.α -acetyltransferase protein isencoded by an open reading frame of 2562 bases and consists of 854 aminoacids. Its molecular weight calculated from its amino acid compositionis 98,575 daltons, and this molecular weight agrees with the subunitM_(r), estimated to be 95,000±2,000. As described above, the proteinsequence analysis of the native protein revealed it to be N-terminallyblocked, it is likely that after the cleavage of N-terminal Met residuethat the penultimate seryl residue was acylated (possibly acetylated).Although the enzyme is not known to be a glycoprotein, it contains 6putative N-glycosylation sites (i.e., Asn-X-Ser (or Thr) sequences) atresidues 120-122, 161-163, 643-645, 702-704, 761-763, 792-793. Theextended, hydrophilic region between residues 508 and 720 is an unusualstructural feature of the molecule, although it is not clear whetherthis region plays a functional role in the regulation or localization ofthe enzyme. A comparison of the protein sequences of N.sup.α-acetyltransferase to other acetyltransferases does not reveal anappreciable percent similarity between them, although certain shortsequences have a greater than 50% similarity. These are likely regionswhere site-specific mutations should be introduced in early attempts toidentify residues involved in catalysis.

Taken together, the Northern and Southern blots indicate that there isone gene encoding this N.sup.α -acetyltransferase. However, it is notclear whether or not yeast contains still other acetyltransferasescapable of modifying the α-NH₂ group of proteins. Further, previousstudies on the substrate specificity of the yeast N.sup.α-acetyltransferase have clearly demonstrated that this enzyme is notcapable of acetylating ε-NH₂ groups in peptide substrates or inhistones, although a histone-specific acetyltransferase has beendemonstrated in yeast (Travis, G. H., J. Biol. Chem. 259:14406-14412(1984)).

The AAA1 gene is located on chromosome 4 and is positioned immediatelyadjacent to the 5' flanking sequence of the SIR2 gene. Since SIR2 andthree other unlinked SIR gene affect trans repression of thetranscription of the HMR and HML genes, which are involved indetermining the mating type of haploid yeast, there is no clear-cutrelationship between the function of these genes and AAA1.

The yeast AAA1 gene will allow the molecular details of the role N.sup.α-acetylation in the sorting and degradation of eukaryotic proteins to bedetermined.

Cloning of N.sup.α -acetyltransferase.

This invention further comprises the amino acid sequences of N.sup.α-acetyltransferase, the genetic sequences coding for the enzyme,vehicles containing the genetic sequence, hosts transformed therewith,enzyme production by transformed host expression, and utilization of theenzyme in the acetylation of peptides or proteins.

The DNA sequence coding for N.sup.α -acetyltransferase may be derivedfrom a variety of sources. For example, mRNA encoded for N.sup.α-acetyltransferase may be isolated from the tissues of any species thatproduces the enzyme, by using the Northern blot method (Alwine et al.,Method Enzymol. 68:220-242 (1979)), and labeled oligonucleotide probes.The mRNA may then be converted to cDNA by techniques known to thoseskilled in the art. The probes may be synthesized based on the knownamino acid sequence of N.sup.α -acetyltransferase as described above.

Alternately, degenerative DNA probes maybe used to screen a DNA libraryof a species that produces N.sup.α -acetyltransferase, thereby isolatinga clone that contains the DNA sequence encoding the enzyme. The DNAlibrary is created by the fragmentation, using one or more restrictionendonucleases of the genomic DNA, followed by incorporation intovectors, and use thereof to transform host cells, which are then platedand screened.

The DNA probe may be labeled with a detectable group. Such detectablegroup can be any material having a detectable physical or chemicalproperty. Such materials have been well-developed in the field ofimmunoassays and in general most any label useful in such methods can beapplied to the present invention. Particularly useful are enzymaticallyactive groups, such as enzymes (see Clin. Chem. 22:1243 (1976)) enzymesubstrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Pat.Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Pat. No.4,134,792); fluorescers (see Clin. Chem. 25:353 (1979)); chromophores;luminescers such as chemiluminescers and bioluminescers (see Clin. Chem.25:512 (1979)); specifically bindable ligands; proximal interactingpairs; and radioisotopes such as ³ H, ³⁵ S, ³² P, ¹²⁵ I and ¹⁴ C. Suchlabels and labeling pairs are detected on the basis of their ownphysical properties (e.g., fluorescers, chromophores and radioisotopes)or their reactive or binding properties (e.g., enzymes, substrates,coenzymes and inhibitors). For example, a cofactor-labeled probe can bedetected by adding the enzyme for which the label is a cofactor and asubstrate for the enzyme. For example, one can use an enzyme which actsupon a substrate to generate a product with a measurable physicalproperty. Examples of the latter include, but are not limited to,beta-galactosidase, alkaline phosphatase and peroxidase.

A DNA sequence encoding N.sup.α -acetyltransferase may be recombinedwith vector DNA in accordance with conventional techniques, includingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide appropriate termini, filling in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and ligation with appropriate ligases.

To express N.sup.α -acetyltransferase transcriptional and translationalsignals recognized by an appropriate host element are necessary.Eukcaryotic hosts may be mammalian cells capable of culture in vitro,particularly leukocytes, more particularly myeloma cells or othertransformed or oncogenic lymphocytes, e.g., EBV-transformed cells.Alternatively, non-mammalian cells may be employed, such as bacteria,fungi, e.g., yeast, filamentous fungi, or the like.

Possible hosts for N.sup.α -acetyltransferase, production are mammaliancells, grown in vitro in tissue culture or in vivo in animals. Mammaliancells may provide post-translational modifications to N.sup.α-acetyltransferase molecules including correct folding or glycosylationof the correct sites. Mammalian cells which may be useful as hostsinclude cells of fibroblast origin such as VERO or CHO-K1, or cells oflymphoid origin, such as the hybridoma SP2/O-AG14 or the myelomaP3×63Sgh, and their derivatives. Usually the N.sup.α -acetyltransferaseconstruct will be part of a vector having a replication systemrecognized by the host cell.

In one embodiment, a procaryotic cell is transformed by a plasmidcarrying the N.sup.α -acetyltransferase-encoded gene. Bacterial hosts ofparticular interest include E. coli K12 strain 294 (ATCC 31446), E. coliX1776 (ATCC 31537), E. coli W3110 (F⁻, lambda⁻, phototropic (ATCC27325)), and other enterobacterium such as Salmonella tyohimurium orSerratia marcescens, and various Pseudomona species. Under suchconditions, the N.sup.α -acetyltransferase will not be glycosylated. Theprocaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

In general, such vectors containing replicon and control sequences whichare derived from species compatible with a host cell, are used inconnection with the host. The vector ordinarily carries a replicon site,as well as specific genes which are capable of providing phenotypicselection in transformed cells. The expression of the N.sup.α-acetyltransferase-encoded DNA can also be placed under control of otherregulatory sequences which may be homologous to the organism in itsuntransformed state. For example, lactose-dependent E. coli chromosomalDNA comprises a lactose or lac operon which mediates lactose utilizationby elaborating the enzyme β-galactosidase. The lac control elements maybe obtained from bacteriophage lambda plac5, which is infective for E.coli. The lac promoter-operator system can be induced by IPTG.

Other promoter/operator systems or portions thereof can be employed aswell. For example, colicin E1, galactose, alkaline phosphatase,tryptophan, xylose, tax, and the like can be used.

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSB40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or markers which allow selection of host cellswhich contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper or the like. The selectablemarker gene can either be directly linked to the DNA sequences to beexpressed, or introduced into the same cell by co-transformation.Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements include those described by Okayama,H., Mol. Cel. Biol. 3:280 (1983), and others.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending on the nature of the host. Thetranscriptional and translational signals may be derived from viralsources, such as adenovirus, bovine papilloma virus, simian virus, orthe like, where the regulatory signals are associated with a particulargene which has a high level of expression. Alternatively, promoters frommammalian expression products, such as actin, collagen, myosin, etc.,may be employed. Transcriptional initiation signals may also be selectedwhich allow for repression or activation, so that expression of thegenes may be modulated. Of interest are regulatory signals which aretemperature-sensitive so that varying the temperature, expression can berepressed or initiated, or are subject to chemical regulation, e.g.,metabolite.

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced to anappropriate host. Various techniques may be employed, such as protoplastfusion, calcium phosphate precipitation, electroporation or otherconventional techniques. After the fusion, the cells are grown in mediaand screened for appropriate activities. Expression of the gene(s)results in production of the N.sup.α -acetyltransferase.

The host cells for N.sup.α -acetyltransferase production may beimmortalized cells, primarily myeloma or lymphoma cells. These cells maybe grown in an appropriate nutrient medium in culture flasks or injectedinto a synergistic host, e.g., mouse or rat, or immunodeficient host orhost site, e.g., nude mouse or hamster pouch.

The N.sup.α -acetyltransferase of the invention may be isolated andpurified in accordance with conventional conditions, such as extraction,precipitation, chromatography, affinity chromatography, electrophoresis,or the like.

IV. Uses of N.sup.α -Acetyltransferase.

N.sup.α -acetyltransferase, once produced and purified, can be used, forexample, in a pharmaceutical manufacturing environment to N-acetylate apeptide or protein. The N-acetylation is useful in reducing degradationof proteins to be used therapeutically. See the discussion following A.Klibinov, "Unconventional Catalytic Properties of Conventional Enzymes,"in Basic Biology of New Developments in Biotechnology, pp. 497-518 (A.Hollaender, ed. 1973) On the use of enzymes.

Expression of the N.sup.α -acetyltransferase in Plants.

Further, the N.sup.α -acetyltransferase can be introduced into a plantby genetic engineering techniques to enhance the rate of acetylation. Itis known that certain herbicides are inactivated by acetylation.Therefore, it is possible to produce a plant that is moreherbicide-tolerant. In thus another embodiment of this invention, theN.sup.α -acetyltransferase gene is used to transform a plant to enhancethe herbicidal tolerance of the plant.

The coding region for a N.sup.α -acetyltransferase gene that may be usedin this invention may be homologous or heterologous to the plant cell orplant being transformed. It is necessary, however, that the geneticsequence coding for N.sup.α -acetyltransferase be expressed, andproduced, as a functional protein or polypeptide in the resulting plantcell. Thus, the invention comprises plants containing either homologousN.sup.α -acetyltransferase genes or heterologous N.sup.α-acetyltransferase genes that express the enzyme.

In one embodiment of this invention, the N.sup.α -acetyltransferasecomprises a plant N.sup.α -acetyltransferase that is homologous to theplant to be transformed. In another embodiment of this invention, theN.sup.α -acetyltransferase comprises an enzyme that is heterologous tothe plant to be transformed. Moreover, DNA from both genomic DNA andcDNA encoding a N.sup.α -acetyltransferase gene may be used in thisinvention. Further, a N.sup.α -acetyltransferase gene may be constructedpartially of a cDNA clone and partially of a genomic clone. In addition,the DNA coding for the N.sup.α -acetyltransferase gene may compriseportions from various species.

There are a variety of embodiments encompassed in the broad concept ofthe invention. In one of its embodiments, this invention compriseschimeric genetic sequences:

(a) a first genetic sequence coding for a N.sup.α -acetyltransferasethat upon expression of the gene in a given plant cell is functional forN.sup.α -acetyltransferase;

(b) one or more additional genetic sequences operably linked on eitherside of the N.sup.α -acetyltransferase coding region. These additionalgenetic sequences contain sequences for promoter(s) or terminator(s).The plant regulatory sequences may be heterologous or homologous to thehost cell.

In a preferred embodiment, the promoter of the N.sup.α-acetyltransferase gene is used to express the chimeric geneticsequence. Other promoters that may be used in the genetic sequenceinclude nos, ocs, and CaMV promoters. An efficient plant promoter thatmay be used is an overproducing plant promoter. This promoter inoperable linkage with the genetic sequence for N.sup.α-acetyltransferase should be capable of promoting expression of saidN.sup.α -acetyltransferase such that the transformed plant has increasedtolerance to a herbicide. Overproducing plant promoters that may be usedin this invention include the promoter of the small subunit (ss) of theribulose-1,5-biphosphate carboxylase from soybean (Berry-Lowe et al., J.Molecular and App. Gen., 1:483-498 (1982)), and the promoter of thechlorophyll a/b binding protein. These two promoters are known to belight induced in eukaryotic plant cells (see, for example, GeneticEngineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum,New York 1983, pages 29-38; Corruzi, G. et al., J. of Biol. Chem., 258:1399 (1983); and Dunsmuir, P. et al., J. of Mol. and Applied Genet., 2:285 (1983)).

Further, in another preferred embodiment, the expression of the chimericgenetic sequence comprising the N.sup.α -acetyltransferase gene isoperably linked in correct reading frame with a plant promoter and witha gene secretion signal sequence.

The chimeric genetic sequence comprising a N.sup.α -acetyltransferasegene operably linked to a plant promoter, and in the preferredembodiment with the secretion signal sequences, can be ligated into asuitable cloning vector. In general, plasmid or viral (bacteriophage)vectors containing replication and control sequences derived fromspecies compatible with the host cell are used. The cloning vector willtypically carry a replication origin, as well as specific genes that arecapable of providing phenotypic selection markers in transformed hostcells, typically resistance to antibiotics. The transforming vectors canbe selected by these phenotypic markers after transformation in a hostcell.

Host cells that may be used in this invention include prokaryotes,including bacterial hosts such as E. coli. S. typhimurium, and Serratiamarcescens. Eukaryotic hosts such as yeast or filamentous fungi may alsobe used in this invention.

The cloning vector and host cell transformed with the vector are used inthis invention typically to increase the copy number of the vector. Withan increased copy number, the vectors containing the N.sup.α-acetyltransferase gene can be isolated and, for example, used tointroduce the chimeric genetic sequences into the plant cells. Thegenetic material contained in the vector can be microinjected directlyinto plant cells by use of micropipettes to mechanically transfer therecombinant DNA. The genetic material may also be transferred into theplant cell by using polyethylene glycol which forms a precipitationcomplex with the genetic material that is taken up by the cell.(Paszkowski et al., EMBO J. 3:2717-22 (1984)).

In an alternative embodiment of this invention, the N.sup.α-acetyltransferase gene may be introduced into the plant cells byelectroporation. (Fromm et al., "Expression of Genes Transferred intoMonocot and Dicot Plant Cells by Electroporation," Proc. Nat'l. Acad.Sci. U.S.A. 82:5824 (1985)). In this technique, plant protoplasts areelectroporated in the presence of plasmids containing the N.sup.α-acetyltransferase genetic construct. Electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof the plasmids. Electroporated plant protoplasts reform the cell wall,divide, and form plant callus. Selection of the transformed plant cellswith the expressed N.sup.α -acetyltransferase can be accomplished usingthe phenotypic markers as described above.

Another method of introducing the N.sup.α -acetyltransferase gene intoplant cells is to infect a plant cell with Aqrobacterium tumefacienstransformed with the N.sup.α -acetyltransferase gene. Under appropriateconditions known in the art, the transformed plant cells are grown toform shoots, roots, and develop further into plants. The N.sup.α-acetyltransferase genetic sequences can be introduced into appropriateplant cells, for example, by means of the Ti plasmid of Agrobacteriumtumefarians. The Ti plasmid is transmitted to plant cells on infectionby Agrobacterium tumefaciens and is stably integrated into the plantgenome. (Horsch et al., "Inheritance of Functional Foreign Genes inPlants," Science 233:496-498 (1984); Fraley et al., Proc. Nat'l Acad.Sci. U.S.A. 80:4803 (1983).)

Ti plasmids contain two regions essential for the production oftransformed cells. One of these, named transfer DNA (T DNA), inducestumor formation. The other, termed virulent region, is essential for theformation but not maintenance of tumors. The transfer DNA region, whichtransfers to the plant genome, can be increased in size by the insertionof the enzyme's genetic sequence without its transferring ability beingaffected. By removing the tumorcausing genes so that they no longerinterfere, the modified Ti plasmid can then be used as a vector for thetransfer of the gene constructs of the invention into an appropriateplant cell.

All plant cells which can be transformed by Agrobacterium and wholeplants regenerated from the transformed cells can also be transformedaccording to the invention so to produce transformed whole plants whichcontain the transferred N.sup.α acetyltransferase gene.

There are presently two different ways to transform plant cells withAgrobacterium:

(1) co-cultivation of Agrobacterium with cultured isolated protoplasts,or

(2) transforming cells or tissues with Agrobacterium.

Method (1) requires an established culture system that allows culturingprotoplasts and plant regeneration from cultured protoplasts.

Method (2) requires (a) that the plant cells or tissues can betransformed by Agrobacterium and (b) that the transformed cells ortissues can be induced to regenerate into whole plants. In the binarysystem, to have infection, two plasmids are needed: a T-DNA containingplasmid and a vir plasmid.

After transformation of the plant cell or plant, those plant cells orplants transformed by the Ti plasmid so that the enzyme is expressed,can be selected by an appropriate phenotypic marker. These phenotypicalmarkers include, but are not limited to, antibiotic resistance. Otherphenotypic markers are known in the art and may be used in thisinvention.

All plants from which protoplasts can be isolated and cultured to givewhole regenerated plants can be transformed by the present invention sothat whole plants are recovered which contain the transferred N.sup.α-acetyltransferase gene. Some suitable plants include, for example,species from the genera Fragaria, Lotus, Medicago, Onobrychis,Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus,Arabidopsis, Brassira, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium,Zea, Triticum, Sorghum, and Datura.

There is an increasing body of evidence that practically all plants canbe regenerated from cultured cells or tissues, including but not limitedto all major cereal crop species, sugarcane, sugar beet, cotton, fruitand other trees, legumes and vegetables. Limited knowledge presentlyexists on whether all of these plants can be transformed byAgrobacterium. Species which are a natural plant host for Agrobacteriummay be transformable in vitro. Monocotyledonous plants, and inparticular, cereals and grasses, are not natural hosts to Agrobacterium.Attempts to transform them using Agrobacterium have been unsuccessfuluntil recently. (Hooykas-Van Slogteren et al., Nature 311:763-764(1984).) There is growing evidence now that certain monocots can betransformed by Agrobacterium. Using novel experimental approaches thathave now become available, cereal and grass species may betransformable.

Additional plant genera that may be transformed by Agrobacterium includeIpomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus,Santalum, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus, andPisum.

Plant regeneration from cultural protoplasts is described in Evans etal., "Protoplast Isolation and Culture," in Handbook of Plant CellCulture 1:124-176 (MacMillan Publishing Co., New York, 1983); M. R.Davey, "Recent Developments in the Culture and Regeneration of PlantProtoplasts," Protoplasts, 1983--Lecture Proceedings, pp. 19-29(Birkhauser, Basel, 1983); P. J. Dale, "Protoplast Culture and PlantRegeneration of Cereals and Other Recalcitrant Crops," in Protoplasts1983 --Lecture Proceedings, pp. 31-41 (Birkhauser, Bagel, 1983); and H.Binding, "Regeneration of Plants," in Plant Protoplasts, pp. 21-37 (CRCPress, Boca Raton, 1985).

Regeneration varies from species to species of plants, but generally asuspension of transformed protoplasts containing multiple copies of theN.sup.α -acetyltransferase gene is first provided. Embryo formation canthen be induced from the protoplast suspensions, to the stage ofripening and germination as natural embryos. The culture media willgenerally contain various amino acids and hormones, such as auxin andcytokinins. It is also advantageous to add glutamic acid and proline tothe medium, especially for such species as corn and alfalfa. Shoots androots normally develop simultaneously. Efficient regeneration willdepend on the medium, on the genotype, and on the history of theculture. If these three variables are controlled, then regeneration isfully reproducible and repeatable.

The mature plants, grown from the transformed plant cells, are selfed toproduce an inbred plant. The inbred plant produces seed containing thegene for the increased N.sup.α -acetyltransferase. These seeds can begrown to produce plants that have enhanced rate of acetylation.

The inbreds according to this invention can be used to develop herbicidetolerant hybrids. In this method, a herbicide tolerant inbred line iscrossed with another inbred line to produce the hybrid.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are covered by the inventionprovided that these parts comprise the herbicidal tolerant cells.Progeny and variants, and mutants of the regenerated plants are alsoincluded within the scope of this invention.

In diploid plants, typically one parent may be transformed by theN.sup.α -acetyltransferase genetic sequence and the other parent is thewild type. After crossing the parents, the first generation hybrids (F1)will show a distribution of 1/2 N.sup.α -acetyltransferase/wild type:1/2N.sup.α -acetyltransferase/wild type. These first generation hybrids(F1) are selfed to produce second generation hybrids (F2). The geneticdistribution of the F2 hybrids are 1/4N.sup.α -acetyltransferase/n.sup.α-acetyltransferase:1/2 N.sup.α -acetyltransferase/wild type:1/4 wildtype/wild type. The F2 hybrids with the genetic makeup of N.sup.α-acetyltransferase/N.sup.α -acetyltransferase are chosen as theherbicidal tolerant plants.

As used herein, variant describes phenotypic changes that are stable andheritable, including heritable variation that is sexually transmitted toprogeny of plants, provided that the variant still comprises aherbicidal tolerant plant through enhanced rate of acetylation. Also, asused herein, mutant describes variation as a result of environmentalconditions, such as radiation, or as a result of genetic variation inwhich a trait is transmitted meiotically according to well-establishedlaws of inheritance. The mutant plant, however, must still exhibit aherbicidal tolerance through enhanced rate of acetylation as accordingto the invention.

Having now generally described this invention, the same will be betterunderstood by reference to specific examples, which are included hereinfor purposes of illustration only, and are not intended to be limitingunless otherwise specified.

EXAMPLE 1 ISOLATION AN PURIFICATION OF N.sup.α -ACETYLTRANSFERASEMaterials and Methods

Enzyme Assays

Enzyme samples of 1-10 μl were added to 1.5 ml Eppendorf tubescontaining a reaction mixture of 50 mM Hepes-K⁺, pH 7.4, 150 mM KCl, 1mM DTT, 25 μM [³ H] acetyl coenzyme A (0.5 μCi, [³ H] acetyl coenzyme Afrom Amersham unlabeled acetyl coenzyme A from P-L Biochemicals) and 50μM ACTH (1-24) with a final volume of 100 μl. The assay mixture wasincubated at 30° C. for 30 minutes. The reaction was stopped by adding17 μl 0.5 M acetic acid and chilled in an ice bath. The reaction sampleswere filtrated through 2.5 cm diameter SP membrane (Cuno Inc.) which hadbeen pre-swollen in a wash with 0.5 M acetic acid. The membranes werewashed three times in 1 ml of 0.5 M acetic acid to remove the unreacted[³ H] acetyl coenzyme A. The partial dried membranes were counted on aBeckman LS 3801 scintillation counter. The radioactivity in the controlrepresented acetylation of endogenous compounds was subtracted from eachsample determination. One unit of activity was defined as 1 pmol ofacetyl residues incorporated into ACTH (1-24) under standard assayconditions.

Purification of N.sup.α -Acetyltransferase from Yeast Cell Growth andStorage

Cells of yeast strain TD 71.8 were grown aerobically at 30° C. in YPDmedium (1% yeast extract, 2% Bacto-peptone, 2% glucose), harvested atlate log phase, and stored at 20° C. with 10% (v/v) glycerol for up to 4months without loss of activity.

Cell Extract

Cells were thawed and collected by centrifugation at 4000 rpm for 10minutes (Beckman, JS-4.0 rotor). The cells (800 g, wet weight) wereresuspended in 1 liter of buffer A (1 M sorbitol, 50 mM Tris-HCl, pH7.8, 10 mM MgCl₂, 3 mM DTT) containing 80 mg of lyticase (Sigma) and thecell suspension was shaken very gently at 30° C. for 45 minutes. Allsubsequent steps were carried out at 4° C. The spheroplasts werecollected by centrifugation at 4000 rpm for 10 minutes, washed by gentleresuspension in 500 ml of Buffer A, collected by centrifugation andresuspended gently in 400 ml of Buffer B (10 mM HEPES-K⁺, pH 7.4, 1.5 mMMgCl₂, 10 mM KCl, and 0.5 mM DTT). The spheroplasts were broken up inthis hypotonic buffer by thirty strokes with a glass Dounce homogenizerand 2.0 M cold KCl was added to give a final KCl concentration of 0.2 M.The homogenate was gently shaken for 30 minutes and debris was removedby centrifugation at 14,000 rpm for 45 minutes (Beckman, JA 14 rotor).The supernatant solution was concentrated by ultrafiltration with PM-30membrane (Amicon, Lexington, MA) and dialyzed overnight against 8 litersof HDG buffer (20 mM HEPES-K⁺, pH 7.4, 0.5 mM DTT, 10% (v/v) glyceroland 0.02% NaN³) containing 0.2 M KCl.

DEAE-Sepharose CL-6B Chromatography

DEAE Sepharose CL-6B (Pharmacia, P-L Biochemicals) was prepared,degassed, and packed into a column (55×2.5 cm) following themanufacturer's recommendation. The column was washed with 4 columnvolume of HDG buffer containing 0.2 M KCl (for 0.2 M KCl chromatography)or 50 mM KCl (for linear KCl gradient chromatography). The dialyzedsupernatant fluid was loaded onto DEAE Sepharose CL-6B pre-equilibratedin HDG buffer containing 0.2 M KCl. Acetyltransferase activity waseluted with same buffer at 24 ml/h. The fractions containingacetyltransferase activity were pooled and concentrated to a volume of50 ml using a PM-30 ultrafiltration membrane. This concentrated eluatewas dialyzed overnight against 4 liters of HDG buffer containing 50 mMKCl and then loaded onto DEAE Sepharose CL-6B pre-equilibrated in HDGbuffer containing 50 mM KCl. The column was developed with a lineargradient between 0.05 M (250 ml) and 0.5 M (250 ml) KCl in HDG buffer at24 ml/h and fractions containing acetyltransferase activity were pooledand concentrated to a volume of 5 ml.

Hydroxylapatite Chromatography

The concentrated eluate was dialyzed overnight against 4 liters of 0.05M potassium phosphate buffer, pH 7.4, 0.5 mM DTT, 10% (v/v) glycerol,0.02% NaN₃ and applied to a hydroxylapatite column (2.5×40 cm)pre-equilibrated with dialysis buffer. The column was developed with alinear gradient between 0.05 M (200 ml) and 0.5 M (200 ml) potassiumphosphate buffer, pH 7.4, each in 0.5 mM DTT, 10% (v/v) glycerol, 0.02%NaN₃ and fractions containing acetyltransferase activity were pooled andconcentrated to a volume of 2.5 ml.

DE-52 Chromatography

This concentrated eluate was dialyzed overnight against 4 liters of HDGbuffer containing 50 mM KCl and then loaded onto DE-52 (Whatman) column(2.5×55 cm) pre-equilibrated in HDG buffer containing 50 mM KCl. Thecolumn was developed with a linear gradient between 0.05 M (250 ml) and0.5 M (250 ml) KCl each in HDG buffer at 24 ml/h and fractionscontaining acetyltransferase activity were pooled and concentrated to avolume of 1 ml.

Affi-Gel Blue gel Chromatography

This concentrated eluate was dialyzed overnight against 4 liters of HDGbuffer containing 50 mM KCl and then loaded onto Affi-Gel Blue gelcolumn (1.5×25 cm) (Bio-Rad) pre-equilibrated in HDG buffer containing50 mM KCl. The column was developed with a linear gradient between 0.05M (150 ml) and 1 M (150 ml) KCl each in HDG buffer at 12 ml/h andfractions containing acetyltransferase activity were pooled andconcentrated.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and GelFiltration

Samples of purified acetyltransferase were loaded onto SDS-8%polyacrylamide gels and subjected to electrophoresis, under reducingconditions, as described by Laemmli, U. K., Nature 227:680-685 (1970).For determination of molecular weight, samples of myosin (205,000),β-galactosidase (116,000), phosphorylase (97,400), bovine albumin(66,000), egg albumin (45,000) and carbonic anhydrase were loaded inparallel lanes. Protein bands were stained with Coomassie Brilliant BluR in methol-acetic acid. Determination of apparent molecular weight bygel filtration was performed using a Sepharose CL-4B column (2.5×96 cm).The elution buffer was HDG buffer containing 0.2 M KCl and the flow ratewas 20 ml/h. The elution volume of acetyltransferase was determined bythe standard assay and the apparent molecular weight calculated from therelative elution volumes of protein standards including thyroglobulin(669,000), apoferritin (443,000), β-amylase (200,000), alcoholdehydrogenase (150,000), albumin (66,000) and carbonic anhydrase(29,000).

Amino Acid and Protein Sequence Analysis

The amino acid composition was obtained using a Beckman 6300 Amino AcidAnalyzer after 24 hr hydrolysis at 110° C. in 6 N HCl containing 0.1%phenol. Protein sequence analysis were carried out using an AppliedBiosystems 470A Protein Sequencer and an Applied Biosystems 120A PthAnalyzer.

EXAMPLE 2 MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING N.sup.α-ACETYLTRANSFERASE FROM SACCHAROMYCES CEREVISIAE Materials and MethodsProtein Sequence Analysis of N.sup.α -acetyltransferase.

N.sup.α -acetyltransferase was purified from yeast as previouslydescribed above. N.sup.α -acetyltransferase (3 nmoles) was reduced andalkylated, precipitated with cold chloroform/methanol, redissolved in0.1 M NH₄ HCO₃, incubated with TPCK-treated trypsin (EC 3.4.21.4; CopperBiomedical, Malvern, PA) (120 pmol) for 24 hr at 37° C., recovered bylyophilization, and dissolved in 6 M guanidine hydrochloride in 0.1% CF₃COOH for HPLC.

Tryptic peptides were separated on a Vydac phenyl (0.46×25 cm) HPLCcolumn, and selected fractions were rechromatographed isocratically onceor twice (Wong, W. W., Proc. Natl. Acad. Sci. USA 82:7711-7715 (1985)).Chromatographic peaks were detected at 214 and 280 nm, collectedmanually, and lyophilized. The tryptic peptides were sequenced byautomated Edman degradation performed with an Applied Biosystems 470AProtein Sequencer and an Applied Biosystems 120 Pth Analyzer (Moore, S.,In: Chemistry and Biology of Peptides, Meienhofer, J. (ed.), Ann ArborScience, Ann Arbor, MI, pp. 629-652 (1972)).

Construction and Screening of cDNA Library.

Yeast RNA was isolated as described by Sherman et al. (Sherman, F., etal., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, ColdSpring Harbor, NY (1986)). Poly(A)⁺ RNA was selected onoligo(dT)-cellulose (Aviv, H., et al., Proc. Natl. Acad. Sci. USA69:1408-1412 (1972)). cDNA was synthesized from 10 μg of poly(A)⁺ RNA bythe method of Okayama and Berg (Okayama, H., et al., Mol. Cell Biol.2:161-170 (1982)), as modified by Gubler and Hoffman (Gubler, U., etal., Gene 25:263-269 (1983)), except that 10% of second strand was [³²P]-labeled. The cDNA was prepared for ligation to λgt11 arms using amethod introduced by Dr. Brian Seed, Department of Molecular Biology,Massachusettes General Hospital. After the ends of the cDNA were madeblunt with T4 DNA polymerase, the cDNA was ligated to adaptorsconsisting of two oligonucleotides: 3' CTCTAAAG 5' and 5' ACACGAGATTTC3'. This cDNA was fractionated on a 5 to 20% linear KOAc gradient (5 ml)using a Beckman SW55 rotor centrifuged for 3 hr at 50,000 rpm at 22° C.Fractions (0.5 ml) were collected from the bottom of the tube. The cDNAwas precipitated by addition of ethanol and linear polyacrylamide (20μg/ml). The size of the cDNAs in each fraction was determined on a 1%agarose gel, and the fractions containing cDNAs between 1 and 8 kb werepooled. Ten micrograms of λgt11 DNA (Young, R. A., et al., Proc. Natl.Acad. Sci. USA 80:1194-1198 (1983)) was digested with EcoRI and ligatedto adaptors (3' GTGTGACCAGATCTCTTAA 5' and 5' CTGGTCTAGAG 3') andprecipitated with PEG8000. 600 ng of λgt11 DNA bearing adaptors wasligated to 150 ng of size-selected cDNA bearing complementary adaptorsin 2 μl and packaged in vitro (Maniatis, T., et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY (1982)) (Stratagene). Escherichia coli strain Y1088 was infected withrecombinant phage, and the library was amplified once. The recombinantfrequency was approximately 82%.

Among several peptide sequences, two peptides (peptides 27-3 and 11-3-2;FIG. 12A) were chosen for constructing two oligonucleotide probes (N1and N2) based on most probable codon usage (Lathe, R., J. Mol. Biol.183:1-12 (1985)). The oligonucleotide probes were synthesized with anApplied Biosystems 380A DNA synthesizer by using the silica-basedsolid-phase method (Matteucci, M. D., J. Am. Chem. Soc. 103:3185-3191(1981)) and proton-activated nucleoside phosphoramidite method(Beaucage, S. L., Tetrahedron Lett. 22:1859-1862 (1981)). The purifiedoligonucleotide were isolated from the crude synthetic mixtures by PAGEand labeled to a specific activity of 2-8×10⁸ cpm/μg by using [λ-³²P]-ATP (New England Nuclear) and T4 polynucleotide kinase (New EnglandBiolabs) (Zoeller, M., et al., DNA 3:479-488 (1985)).

In the initial screen, 500,000 recombinant clones in λgt11 yeast cDNAlibrary were plated on E. coli Y1088. Duplicate transfers of the cloneswere made onto nitrocellulose, and the filters were prepared forhybridization (Zoeller, M., et al., DNA 3:479-488 (1985)). Afterward,the filters were washed twice at room temperature in 6×SSC (0.15 MNaCl/15 mM sodium citrate (NaCl/Cit) containing 0.1% SDS and 0.05%NaPPi), washed once at 5° C. below the minimum t_(d) (temperature ofprobe dissociation based on G/C content), and exposed on x-ray film for2 to 4 days. Maximum and minimum t_(d) were determined for two pools ofredundant oligonucleotide probes (N3 and N4) (Suggs, S. V., et al., In:Developmental Biology Using Purified Genes, Brown, D. (ed.), AcademicPress, New York, pp. 683-693 (1981)).

DNA Sequencing and Blot Analysis.

cDNA fragments were cleaved out from recombinant λgt11 phase DNA byEcoRI digestion. The cDNA fragments were separated by gelelectrophoresis in low melting point agarose. The correct DNA band wassliced out, the gel was melted at 65° C., and the DNA was extracted withphenol. The purified cDNA fragments were cloned into the Bluescriptplasmid (Stratagene). Both orientations of the complete sequence of theyeast N.sup.α -acetyltransferase (AAA1) gene were determined byexonuclease III deletion (Henikoff, S., Gene 28:351-359 (1984)), thedideoxy chain termination method of Sanger (Sanger, F., et al., J. Mol.Biol. 94:441-448 (1975)) modified for double-stranded sequencing by Guoet al. (Guo, L.-H., et al., Nucl. Acids Res. 11:5521-5539 (1983)), andspecific priming with synthetic oligonucleotides. All restrictionenzymes were purchased from New England Biolabs. RNA and DNA markerswere obtained from Bethesda Research Laboratories. Biotrans nylonmembrane was from ICN. Poly(A)⁺ RNA was analyzed by Northern blothybridization (Lehrach, H., Biochemistry 16:4743-4751 (1977); Thomas, P.S., Proc. Natl. Acad. Sci. USA 77:5201-5202 (1980)). Genomic DNA wasisolated from yeast (Sherman, F., et al., Methods in, Cold Spring HarborLaboratory, Cold Spring Harbor, NY (1986)), restriction enzymedigestion, and analyzed by Southern blot hybridization (Southern, E., J.Mol. Biol. 98:503-517 (1975)). The chromosome bearing the AAAI gene waslocated by hybridizing to a Saccharomyces chromo-di-hybridizer(Clonetech) (i.e., a yeast chromosomal blot).

Analysis of Tryptic Peptides of N.sup.α -Acetyltransferase.

The method adopted for the identification and cloning of cDNA sequencesderived from N.sup.α -acetyltransferase mRNA utilized oligonucleotideprobes that were constructed based on amino acid sequences of purifiedN.sup.α -acetyltransferase tryptic peptides and on codon usage frequencydata (Lathe, R., J. Mol. Biol. 183:1-12 (1985)). Tryptic peptides fromyeast N.sup.α -acetyltransferase were separated by reversed-phase HPLCand collected as 62 pools representing distinct peaks or shoulders (FIG.11). Three nanomoles of purified N.sup.α -acetyltransferase was reduced,alkylated, digested with trypsin, and chromatographed on a 0.46×25 cmVydac phenyl HPLC column with 0.1% CF₃ COOH in a linear gradient of0-60% CH₃ CN over 2 hr. After one or two additional isocratic HPLCseparations to resolve further individual sequenceable tryptic peptides(Wong, W. W. et al., Proc. Natl. Acad. Sci. USA 82:7711-7715 (1985)),the sequences of 16 peptides, comprising approximately 30% of the entireN.sup.α -acetyltransferase molecule were determined.

Synthesis of Oligonucleotide Probes.

Two synthetic, codon-usage based oligonucleotides of 57 bases (N1) and48 bases (N2), corresponding to sequence of a 19-(peptide 27-3) and a16-residue (peptide 11-3-2) peptide, respectively, were used in theinitial screening of the cDNA library (FIG. 12A). In addition, twodegenerate oligonucleotide probes of 23 (5' CCXTTGACYTTYTTRCAAGATAA 3')and 20 (3 TCRTCRTGCATYTGRAARTA 5') bases with 64- and 32-foldredundancy, designated N3 and N4, were synthesized based on sequencedata for peptides 29-1 and 10-3-1, respectively. The use of fouroligonucleotide probes derived from four discrete amino acid sequencesallowed the unequivocal identification of the cDNA clones ending N.sup.α-acetyltransferase. The protein sequence analyses were completed withrepetitive yields between 87% and 93% for 100-200 nmol of each peptide.

Cloning of the Yeast N.sup.α -Acetyltransferase cDNA.

After initial screening of 500,000 recombinant cDNA clones in the yeastλgt11 cDNA library, eleven clones hybridized to both oligonucleotides N1and N2. These clones, designated λN1 to λN11, also hybridized witholigonucleotides N3 and N4, and their cDNA inserts were analyzed byrestriction enzyme digestions and Southern blot analyses. EcoRIdigestion revealed inserts that lacked internal EcoRI sites and rangedfrom 2.0 to 2.7 kb. The six longest cDNA inserts were subcloned as EcoRIfragments into the Bluescript plasmid, and additional restriction enzymemapping, Southern blot analyses and nucleotide sequence analyses werecarried out. All six cDNA clones (pBN1, pBN3, pBN7, pBN9, pBN10, andpBN11) displayed identical restriction maps (FIG. 12B).

Sequence Analysis of the cDNA Clones.

The complete nucleotide sequence, both orientations, of pBN1 wasdetermined using exonuclease III deletions and double-stranded dideoxychain termination method. The protein sequence translated from thesequence of the 2.71 kb cDNA insert of pBN1 contained identicalsequences to those determined from the protein sequence analyses of 16tryptic peptides (FIG. 12C). However, there was a stop codon located atnucleotides 1409-1411, and the putative reading frame was shifted afterthis stop codon. Hence, the nucleotide sequences within thecorresponding region of the other 5 cDNA clones were determined usingsynthetic oligonucleotide primers. These 5 clones each contained anadditional T which maintained an open reading frame. It is evident thatthe termination codon in pBN1 was introduced by deletion of a T atnucleotide 1410, presumably resulting from a lack of fidelity for thereverse transcriptase reaction during cDNA synthesis.

The complete nucleotide sequence of the yeast N.sup.α -acetyltransferasecDNA is shown in FIG. 12C. The translation initiation site is determinedunequivocally, because there is only one ATG codon (nucleotides 22-24),which is preceded by an in-frame termination codon (nucleotides 13-15),located upstream and in-frame with the DNA sequence encoding a trypticpeptide (residues 61-82), which precedes the next methionine in thesequence located at residue 101. There is an open reading frame of 2562nucleotides encoding the 854 amino acid residues of N.sup.α-acetyltransferase, a termination codon (TAG) at nucleotides 2584-2586,and a polyadenylation signal (ATAAAA) located 18 nucleotides upstreamfrom the poly (A) tail.

Comparison of DNA and Protein Sequence Data for Yeast N.sup.α-acetyltransferase cDNA with DNA and Protein Sequence Databases

The EMBL Nucleic Acid Database revealed an 842-base identity between a3' region of the cDNA encoding N.sup.α -acetyltransferase, beginning atnucleotide 1858 and ending at nucleotide 2699, and the genomic DNAsequence upstream of the 5' end of the SIR2 gene located on chromosome 4(unpublished data of Shore, D., et al. (Shore, D., et al., EMBO J.3:2817-2823 (1984)) and deposited in the EMBL Nucleic Acid Database).Comparisons between the protein sequence of yeast N.sup.α-acetyltransferase and choline acetyltransferase from chicken liver(Deguchi, T., et al., J. Biol. Chem. 263:7528-7533 (1988)) revealed apercent similarity of 10%, 12%, 12%, 14%, and 15%, respectively,although there are sequences of 6 to 16 amino acid residues which havepercent similarities between 44% and 83% (Devereux, J., et al., Nucl.Acids Res. 12:387-395 (1984)).

Northern, Southern, and Chromosomal Blot Analyses.

Northern blot analysis of yeast poly(A)⁺ mRNA using a random-primed [³²P]-labeled yeast N.sup.α -acetyltransferase cDNA probe (pBN1) revealed a2.7 kb RNA band. Yeast DNA (10 μg) was digested with indicatedrestriction enzymes. The restriction fragments were electrophoresed in0.8% agarose in Tris-borate buffer. The DNA was transferred onto a nylonmembrane and hybridized with random primed, [³² P]-AAA1 cDNA (derivedfrom pBN1) for 24 hr and washed (Southern, E., J. Mol. Biol. 98:503-517(1975)).

Southern blot analysis of restriction enzyme digested yeast genomic DNArevealed that the sizes of fragments were similar to the sizes for therestriction fragments of yeast N.sup.α -acetyltransferase cDNA (pBN1)(FIG. 12B). Yeast poly(A)⁺ RNA (10 μg) was electrophoresed on a 1.2%agarose/formaldehyde gel (Maniatis, T., et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY(1982)). The mRNA was transferred onto a nylon membrane and hybridizedwith random primed, [³² P]-AAA1 cDNA (derived from pBN1) for 24 hr andwashed (Lehrach, H., Biochemistry 16:4743-4751 (1977); Thomas, P. S.,Proc. Natl. Acad. Sci. USA 77:5201-5202 (1980)). The gel lane containingthe RNA markers was sliced out, visualized by staining with ethidiumbromide, and used for determining the molecular size of the yeastpoly(A)⁺ mRNA.

Chromosomal blot analysis with the probe indicates that the yeastN.sup.α -acetyltransferase gene (AAA1) is located on chromosome IV. Anagarose gel of yeast chromosomal DNA was hybridized with random primed,[³² P]-AAA1 cDNA (derived from pBN1) for 24 hr and washed according tothe manufacturer's recommendations.

Hydrophobicity Profile for Yeast N.sup.α -Acetyltransferase.

The hydrophobicity profile in FIG. 13 was determined using the algorithmof Kyte and Doolittle (Kyte, J., et al., J. Mol. Biol. 157:105-132(1982)) with a window size of 9 (FIG. 13). The protein is rich incharged amino acids, including 96 lysine, 37 arginine, 59 aspartic acid,and 60 glutamic acid residues. In addition, there is an extendedhydrophilic region between residues 508 and 720, which is rich inresidues associated with β-turn conformations (Chou, P. Y., et al., In:Peptides: Proceedings of the Fifth American Peptide Symposium, Goodman,M., et al. (eds.), John Wiley and Sons, New York, pp. 284-287 (1977)).

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those ordinarily skilled in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.

What is claimed is:
 1. A substantially purified N.sup.α-acetyltransferase having the following characteristics:(a) a molecularweight of about 180,000; (b) two subunit peptides having molecularweights of about 95,000 each; (c) pH optimum 9.0; (d) maximum specificactivity at temperature from 30° to 42° C. wherein one unit of specificactivity is defined as 1 pmol of acetyl residues incorporated into asubstrate containing the first 24 amino acid residues ofadrenocorticotropic hormone (ACTH) per mg of protein under standardconditions; (e) inhibited by divalent cations Cu²⁺ and Zn²⁺ ; and (f) anability to acetylate a substrate peptide or protein molecule whereinsaid ability is specifically dependent on the amino-terminal residue ofsaid substrate peptide or protein molecule.
 2. The N.sup.α-acetyltransferase of claim 1 having enzyme specific activity greaterthan 100 wherein one unit of specific activity is defined as 1 pmol ofacetyl residues incorporated into a substrate containing the first 24amino acid residues of adrenocorticotropic hormone (ACTH) per mg ofprotein under standard assay conditions.
 3. The N.sup.α-acetyltransferase of claim 1 having enzyme specific activity greaterthan 300 wherein one unit of specific activity is defined as 1 pmol ofacetyl residues incorporated into a substrate containing the first 24amino acid residues of adrenocorticotropic hormone (ACTH) per mg ofprotein under standard assay conditions.
 4. The N.sup.α-acetyltransferase of claim 1 having enzyme specific activity greaterthan 1000 wherein one unit of specific activity is defined as 1 pmol ofacetyl residues incorporated into a substrate containing the first 24amino acid residues of adrenocorticotropic hormone (ACTH) per mg ofprotein under standard assay conditions.
 5. The N.sup.α-acetyltransferase of claim 1 having enzyme specific activity greaterthan 7000 wherein one unit of specific activity is defined as 1 pmol ofacetyl residues incorporated into a substrate containing the first 24amino acid residues of adrenocorticotropic hormone (ACTH) per mg ofprotein under standard assay conditions.
 6. A method for recovering thesubstantially purified N.sup.α -acetyltransferase of claim 1 from asample containing N.sup.α -acetyltransferase comprising the followingsteps:(a) recovering crude N.sup.α -acetyltransferase from a samplecontaining said N.sup.α -acetyltransferase; (b) subjecting said crudeN.sup.α -acetyltransferase from step (a) to ion exchange chromatographyto obtain active fractions of N.sup.α -acetyltransferase wherein oneunit of specific activity is defined as 1 pmol of acetyl residuesincorporated into a substrate containing the first 24 amino acidresidues of adrenocorticotropic hormone (ACTH) per mg of protein understandard assay conditions: (c) applying said active fractions of N.sup.α-acetyltransferase from step (b) to adsorption hydroxylapatitechromatography to obtain partially purified N.sup.α -acetyltransferase;(d) applying said active fractions of N.sup.α -acetyltransferase fromstep (c) to ion exchange chromatography to obtain a more highly purifiedN.sup.α -acetyltransferase; and (e) purifying N.sup.α -acetyltransferaseto homogeneity by applying said partially purified n.sup.α-acetyltransferase from step (d) to an affinity chromatography andeluting therefrom substantially purified N.sup.α -acetyltransferase. 7.A substantially purified N.sup.α -acetyltransferase with a molecularweight of about 180,000 daltons, said N.sup.α -acetyltransferase beingcomposed of two subunit peptides having molecular weights of about95,000 each, having specific enzyme activity greater than 100 whereinone unit of specific activity is defined as 1 pmol of acetyl residuesincorporated into a substrate containing the first 24 amino acidresidues of adrenocorticotropic hormone (ACTH per mg of protein understandard assay conditions obtainable by a process comprising the stepsof:(a) recovering crude N.sup.α -acetyltransferase from a samplecontaining said N.sup.α -acetyltransferase; (b) subjecting said crudeN.sup.α -acetyltransferase from step (a) to ion exchange chromatographyto obtain active fractions of N.sup.α -acetyltransferase wherein oneunit of specific activity is defined as 1 pmol of acetyl residuesincorporated into a substrate containing the first 24 amino acidresidues of adrenocorticotropic hormone (ACTH) per mg of protein understandard assay conditions: (c) applying said active fractions of N.sup.α-acetyltransferase from step (b) to adsorption hydroxylapatitechromatography to obtain partially purified N.sup.α -acetyltransferase;(d) applying said active fractions of N.sup.α -acetyltransferase fromstep (c) to ion exchange chromatography to obtain a more highly purifiedN.sup.α -acetyltransferase; and (e) purifying N.sup.α -acetyltransferaseto homogeneity by applying said partially purified N.sup.α-acetyltransferase from step (d) to affinity chromatography and elutingtherefrom substantially purified N.sup.α -acetyltransferase.